Abstract

In a previously published insertional mutagenesis screen for candidate brain tumor genes in the mouse using a Moloney mouse leukemia virus encoding platelet-derived growth factor (PDGF)-B, the Sox10 gene was tagged in five independent tumors. The proviral integrations suggest an enhancer effect on Sox10. All Moloney murine leukemia virus/PDGFB tumors had a high protein expression of Sox10 independently of malignant grade or tumor type. To investigate the role of Sox10 in gliomagenesis, we used the RCAS/tv-a mouse model in which the expression of retroviral-encoded genes can be directed to glial progenitor cells (Ntv-a mice). Both Ntv-a transgenic mice, wild-type, and Ntv-a p19Arf null mice were injected with RCAS-SOX10 alone or in combination with RCAS-PDGFB. Infection with RCAS-SOX10 alone did not induce any gliomas. Combined infection of RCAS-SOX10 and RCAS-PDGFB in wild-type Ntv-a mice yielded a tumor frequency of 12%, and in Ntv-a Arf−/− mice the tumor frequency was 30%. This indicates that Sox10 alone is not sufficient to induce gliomagenesis but acts synergistically with PDGFB in glioma development. All induced tumors displayed characteristics of PNET-like structures and oligodendroglioma. The tumors had a strong and widely distributed expression of Sox10 and PDGFR-α. We investigated the expression of Sox10 in other human tumors and in a number of gliomas. The Sox10 expression was restricted to gliomas and melanomas. All glioma types expressed Sox10, and tumors of low-grade glioma had a much broader distribution of Sox10 compared with high-grade gliomas. (Mol Cancer Res 2007;5(9):891–7)

Keywords:

Brain/central nervous system cancers

Tumor promotion and progression

Cancer susceptibility genes

Oncogenes

Viral transformation and carcinogenesis

Introduction

Glioblastoma is the most common and malignant primary brain tumor in the adult, with a mean survival time after diagnosis of 1 year. Primary glioblastoma develops de novo without any known intermediate stages of the tumor, whereas secondary glioblastoma arises by progression from a lower grade to a higher tumor grade. Common genetic alterations in primary glioblastoma are amplification of EGFR and MDM2 and deletions of the INK4A gene (1, 2). During the progression of secondary glioblastoma, mutations accumulate over time and more than 65% of the tumors have mutations in TRP53. Overexpression of the platelet-derived growth factor receptor-α (PDGFRA) and PDGFA are characteristic for secondary glioblastoma (3). Constitutive expression of growth factors and their receptors has been shown to be necessary for the development of brain tumors resulting in autocrine stimulation and increased activity of downstream pathways (4). Inactivation of the tumor suppressor gene phosphatase and tensin homologue (PTEN; refs. 5, 6) is another common trait of glioblastoma. PTEN signals through Akt and many glioblastomas have an increased activity of Akt (7).

To better understand the role of PDGF in gliomagenesis, we have generated a mouse glioma model in which a Moloney murine leukemia virus (MMLV)/PDGFB–containing retrovirus (MMLV/PDGFB) together with a replication-competent helper virus was injected into newborn mouse brains, which resulted in malignant brain tumors (8). All tumors grew invasively and diffusely, and most of them had areas of necrosis and angiogenesis. The tumors stained positively for nestin, suggesting that they had evolved from an immature neuroglial progenitor cell (8). We have postulated that the tumors developed through an autocrine PDGF receptor activation in combination with insertional mutagenesis through proviral integrations. A number of common proviral integration sites were identified, targeting candidate tumor-causing genes (9).

One of the tagged genes was Sox10. Sox10 is a transcription factor and belongs to the Sox superfamily, which all contain a DNA binding motif known as the high-mobility group domain. During development, Sox10 first appears in the developing neural crest and is expressed during the formation of the peripheral nervous system (10). In central nervous system, Sox10 was first found on glial progenitor cells but later also detected in oligodendrocytes in the adult brain (11). Sox10 precedes the expression of PDGFR-α in oligodendrocyte precursors, but once the PDGFR-α is expressed, they are found in the same cells (12). Homozygous Sox10 null mice die before or at birth. The entire peripheral nervous system is defective and motor neurons are absent (13). The levels of PDGFR-α are reduced, suggesting that Sox10 influences the expression of PDGFR-α (12). In human, mutations in the SOX10 gene have been linked to Waardenburg-Shah syndrome type IV, which is characterized by depigmentation of hair and skin, and Hirschsprung's disease (megacolon; ref. 14). Mutations in SOX10 in combination with Waardenburg-Shah syndrome type IV have also been associated with severe dysmyelination syndromes (PCWH; ref. 14). Further, Sox10 is expressed in, and during the development of, melanocytes, which also are derived from the neural crest. During melanocyte specification, Sox10 is responsible for activating the melanocyte transcription regulator Mitf, which controls melanocyte survival and differentiation (15, 16).

In the present investigation, we have studied the role of Sox10 in human and mouse gliomas. For studies in the mouse, we used the RCAS/tv-a model (replication-competent avian leukemia virus splice acceptor/avian leukemia virus receptor) in which the expression of retrovirus-encoded genes can be directed either to glial progenitor cells (Ntv-a mice; mice expressing the tv-a receptor behind the nestin promoter) or astrocytes (Gtv-a mice; mice expressing tv-a by the glial fibrillary acidic protein promoter). We have found that Sox10 has a broad distribution in different types of gliomas in both human and mouse tumors. Sox10 was not able to initiate tumorigenesis by itself, but in combination with an RCAS virus expressing PDGFB, the tumor incidence was increased.

Results

Sox10 Is Highly Expressed in PDGFB-Induced Mouse Gliomas

In our previous screen for candidate glioma genes in the mouse, Sox10 was tagged in five independent MMLV/PDGFB–induced tumors (9). The proviral integration sites were at both transcriptional orientations and located upstream of Sox10 within an area of 60 kb (Fig. 1
). The upstream localization of the proviral insertions suggests an enhancer effect on Sox10.

Schematic illustration of proviral integrations upstream of Sox10. Five proviral integrations were tagged upstream of the transcriptional start site of Sox10 in five different tumors. Arrows illustrate the position and the transcriptional orientation of the proviral genome. Data were adapted from the Ensembl Mouse Genome Browser and exact positions of the integrations can be found in the Retrovirus Tagged Cancer Gene Database (http://RTCGD.ncifcrf.gov/).

The brain tumors induced by the MMLV/PDGFB viruses were of different malignancy grades and could be divided into glioblastoma-like, oligodendroglioma-like, and PNET-like (primitive neuroectodermal tumor) tumors. When analyzing the MMLV/PDGFB tumors for Sox10 expression, we found that all analyzed tumors had a high protein expression of Sox10, independent of the tumor type. The protein was expressed also in tumors without integrations in Sox10 (Fig. 2
). In addition, real-time PCR analysis has previously shown that Sox10 is strongly up-regulated in both early and late MMLV/PDGFB–induced tumors (17).

Sox10 expression in PDGFB-induced tumors. Immunohistochemical staining of Sox10 in mouse gliomas induced with MMLV/PDGFB. A. A representative PNET-like tumor. B. A glioblastoma-like tumor. C. A PNET-like tumor in which one of the Sox10 integrations was identified.

Sox10 Enhances PDGF-Induced Gliomagenesis

To investigate the role of Sox10 in gliomagenesis, we used the RCAS/tv-a mouse model. Cells infected with RCAS virus containing SOX10 (RCAS-SOX10) were injected intracerebrally in newborn mice. Both Ntv-a transgenic mice, wild-type (wt), and Ntv-a p19Arf null mice were injected with RCAS-SOX10 alone or in combination with RCAS-PDGFB (Table 1
). Infection with RCAS-SOX10 alone did not induce any gliomas, neither in Ntv-a wt nor in Ntv-a Arf−/− mice. Injections with RCAS-PDGFB together with an empty RCAS vector (RCAS-X) gave rise to 3 tumors from 37 injected mice in the Ntv-a Arf−/− background, whereas no tumors were detected in Ntv-a wt mice (Table 1). These injections were used as controls for the combined injections of RCAS-SOX10 plus RCAS-PDGFB. The injection of RCAS-SOX10/RCAS-PDGFB in wt Ntv-a mice yielded a tumor frequency of 12%, and in Ntv-a Arf−/− mice the tumor frequency was even higher, 30% (Table 1). When we compared the results of injection of RCAS-SOX10/RCAS-PDGFB with that of RCAS-X/RCAS-PDGFB, we found a significantly higher tumor frequency in both Ntv-a wt and Arf−/− mice (Ntv-a wt: P = 0.0428, RCAS-SOX10/RCAS-PDGFB versus RCAS-X/RCAS-PDGFB; Ntv-a Arf−/−: P = 0.0143, RCAS-SOX10/RCAS-PDGFB versus RCAS-X/RCAS-PDGFB). These results indicated that the Sox10 expression enhanced PDGFB-induced glioma development and that the glioma incidence increased even more when injecting RCAS-SOX10 and RCAS-PDGFB in the Arf−/− background.

Tumor Incidence in Mice Injected with Different RCAS Virus in Different Combinations

The histology of the tumors induced by RCAS-SOX10/RCAS-PDGFB or RCAS-X/RCAS-PDGFB was examined. All the tumors, independent of the presence or absence of p19Arf, displayed morphologic characteristics of PNET-like tumors. All the tumors grew invasively and had vessel formation. The tumor cells were relatively small and displayed perineuronal satellitosis (Fig. 3A
). All tumors showed a strong and widely distributed expression of Sox10 and PDGFR-α (Fig. 3B and C). Only scattered cells in the tumors were positive for the astrocytic marker GFAP (Fig. 3D), suggesting that the main tumor cell population is not of astrocytic origin. The tumors were NG2 positive, indicating a more immature cell of origin (Fig. 3E); NG2 expression in central nervous system is normally found on a class of glia cells with properties of oligodendrocyte type II astrocyte precursor cells (18). Figure 3F shows that the staining for nestin was more or less restricted to the vessels. The tumors also stained positive for one of the earliest expressed transcription factors, Sox2. Sox2 has been suggested to maintain neural cells in an immature and proliferative stage (neural stem cell) both during embryogenesis and in the adult brain (19, 20). The expression of Sox2 and Sox10 were colocalized in most tumor cells but there were also cells positive only for Sox2 or Sox10 (Fig. 3G and H).

Immunohistochemical staining of a RCAS-SOX10– and RCAS-PDGFB–induced PNET-like tumor. Immunohistochemical staining of a representative tumor induced by injection of a combination of RCAS-SOX10 and RCAS-PDGFB in Ntv-a transgenic Arf null mice. A. H&E staining. Arrows, typical perineuronal satellitosis. B. Strong positive staining for Sox10. C. Expression of PDGFR-α. D. Scattered expression of GFAP (i.e., presence of astrocytes in the tumor). E. NG2 expression, indicating that the tumor is of immature nature. F. Nestin, expressed mainly in the vessels. G. Presence of the stem cell marker Sox2. H. Sox10 and Sox2 (yellow) are mainly coexpressed in the tumor cells, but there are cells that only express Sox10 (green) or Sox2 (red).

The Expression of Sox10 in Mouse Gliomas Is High Even in Akt/Kras–Induced Tumors

The high expression of the Sox10 protein both in the MMLV/PDGFB and in the RCAS-SOX10/RCAS-PDGFB–induced tumors prompted us to investigate the expression of Sox10 in other types of experimental mouse glioma. We therefore studied the expression of Sox10 in tumors induced with other oncogenes such as RCAS-Akt and RCAS-Kras, both in combination or alone, and in tumors induced with RCAS-PDGFB using Ntv-a– and Gtv-a–expressing Arf null or Ink−/− mice (Fig. 4
and data not shown). Figure 4A and B shows an Akt/Kras–induced brain tumor in a Gtv-a Arf−/− mouse with a mixed histology containing areas that are glioblastoma- or gliosarcoma-like. In this tumor Sox10 is expressed in the glioblastoma areas but not in the sarcoma areas. Figure 4C and D shows another Akt/Kras–induced tumor with a histology of mixed fibrillar astrocytoma and gliosarcoma. This tumor contains Sox10-positive cells but only in the astrocytoma areas. The tumor depicted in Fig. 4E and F was induced by Akt/Kras in an Ntv-a Arf−/− mouse and has the histologic features of a mixed oligodendroglioma, PNET and gliosarcoma. This tumor was negative for Sox10 expression in the sarcoma areas. Figure 4G and H shows a tumor induced with RCAS-PDGFB in a Gtv-a Arf−/− mouse with a PNET-like histology, which was strongly positive for Sox10.

Sox10 is expressed in mouse glioma induced by Akt and Kras in the RCAS/Tv-a mouse model. A, C, E, and G. H&E staining of the tumor. B, D, F, and H. Corresponding tumor stained for Sox10. The injections are done in Arf null mice. A and B. A tumor induced by the RCAS viruses expressing the oncogenes Akt and Kras. Typical areas of glioblastoma and sarcoma are present. C and D. A fibrillar astrocytoma with sarcoma-like areas induced by injections of RCAS-Akt and RCAS-Kras. E and F. A tumor containing typical areas of oligodendroglioma, PNET, and sarcoma induced by the Akt and Kras viruses. G and H. Typical PNET tumor with perineuronal satellitosis induced by the RCAS-PDGFB virus. The tumors in (E) to (H) contain small immature cells, neuroepithelial-like cells, with blast-like nuclei that are typical for PNET and oligodendroglioma. The tumors in (A), (C), and (G) are induced in G-tva transgenic mice and the tumor in (E) is induced in an Ntv-a transgenic mouse. All tumors are positive for Sox10 but to different extent. It seems like it is the glioma-like parts that are positive for Sox10. S, sarcoma-like areas; GB, glioblastoma-like areas; F, fibrillar areas; vp, vessel proliferation; arrows, perineuronal satellitosis.

All tested mouse gliomas thus expressed Sox10 independent of genetic background or oncogenes used for induction. When evaluating the presence of PDGFRα, we found a regional heterogeneity in the Akt/Ras-induced gliomas. In the Sox10/PDGFB– or PDGFB-induced tumors, the expression of PDGFRα was broader and coincided with a higher expression of Sox10.

Sox10 Expression Is High in Human Low-Grade Gliomas

Because Sox10 is known to be expressed normally in oligodendrocyte precursor cells and in oligodendrocytes (11, 21), we wanted to examine if it is possible to distinguish human oligodendroglioma from astrocytoma based on Sox10 expression. We investigated the expression of Sox10 on a number of human gliomas (Table 2
). Surprisingly, most tumors expressed Sox10 independent of tumor type. Tumors with a low cell density, such as grade 2 astrocytomas and grade 2 oligodendrogliomas, had a broad distribution of Sox10, and grade 3 and grade 4 tumors such as grade 4 glioblastoma, grade 3 oligodendroglioma, and grade 3 ependymoma contained a lower number of Sox10-positive cells (Fig. 5
). The expression of Sox10 in pediatric gliomas showed the same pattern with higher expression in pilocytic astrocytomas (grade 1) compared with glioblastoma (Table 3
). Among the typical child brain tumors, medulloblastoma and PNET, we found detectable staining for Sox10 only in one PNET (no. 4), which was diagnosed as a PNET with astrocytic and neural differentiation. In the remaining PNET tumors, there was no detectable staining and all the medulloblastomas were negative. Both astrocytomas and oligodendrogliomas express Sox10, consistent with the findings in the Akt/Kras mouse tumors where astrocytic areas were also positive for Sox10. Sox10 had a high expression in low-grade gliomas independently of tumor origin both in pediatric and adult gliomas. There are reports (22, 23) suggesting a different cell of origin for medulloblastomas and PNETs compared with gliomas, which could explain the lack of Sox10 expression in these tumors.

Sox10 Expression in Human Tumor Tissue Is Restricted to Glioma and Melanoma

The expression of Sox10 in human tissues and tumors was further analyzed using human tissue microarrays. As expected, Sox10 was expressed in oligodendrocytes in the cerebellum, cortex, lateral ventricles, and hippocampus of the brain (Fig. 5). In the basal portion of epidermis, scattered cells, most likely melanocytes, were positive as well as cells connected to the hair follicles. In salivary gland, the acinic cells most probably were positive. In a few tissues, epithelial cells stained positive; in breast, a subset of the ducts were positive as well as certain ductal glands in respiratory mucosa and in head and neck areas. In pancreas and prostate, a few smooth muscle cells stained positive for Sox10. All other examined tissues were negative or contained only occasionally positive cells.

Analysis of the tumor tissue microarray showed that the malignant gliomas were positive as expected. Sox10 was also very strongly expressed in malignant melanomas as reported (24). Essentially all tumor cells in the 11 tested malignant melanomas were Sox10 positive. Six of 12 basal and squamous skin carcinomas had scattered positive cells, most likely melanocytes. Taking these data together, Sox10 seems to have a restricted distribution pattern both in normal and tumor tissues and expressed mainly in brain/gliomas and in skin/melanoma and basal cell carcinoma.

Discussion

In the previously published series of MMLV/PDGFB–induced brain tumors, five integrations were located upstream of the Sox10, indicating that Sox10 could be a potential oncogene (9). In the present study, we have investigated if Sox10 is able to initiate tumorigenesis on its own or in cooperation with PDGF. We used the RCAS/tv-a system where gene(s) of interest can be expressed in specific cell types in the brain. We found that Sox10 alone is not able to induce gliomagenesis, neither in Ntv-a wt nor in Arf−/− transgenic mice. Although Arf is one of the major suppressor genes in gliomagenesis (25), overexpression of Sox10 in Arf null mice did not initiate gliomagenesis within 12 weeks. However, the RCAS-SOX10 virus significantly enhanced the gliomagenic activity of RCAS-PDGFB in Arf null mice; RCAS-SOX10 caused an almost 4-fold increase in tumor incidence, as compared with RCAS-PDGFB alone. Our data therefore indicate that Sox10 is not an oncogene by itself but may enhance the gliomagenic activity of PDGF in experimental mouse glioma.

Sox10 was uniformly expressed in all mouse gliomas induced by PDGFB vectors, without co-injection with RCAS-SOX10. This finding is in line with the notion that the PDGFB-induced tumors originate from transformed, immature cells of the oligodendrocyte lineage (i.e., oligodendrocyte precursors). This view is further supported by the finding that the tumors induced by PDGFB, plus or minus Sox10, were NG2, PDGFRα, and Sox2 positive; both NG2 and of PDGFRα are markers of oligodendrocyte precursors (18) and Sox2 is a marker of stem cells. Further, the tumors were, to some extent, nestin positive, nestin being another marker for immature cells of the central nervous system (26). One mechanism by which RCAS-SOX10 enhances PDGFB-induced gliomagenesis could therefore be to increase the Sox10-expressing, PDGFB-responsive immature target cell population.

Sox10 expression was higher and more widely distributed in PNET- and oligodendroglioma-like tumors, induced by PDGFB alone or in combination with Sox10, as compared with the tumors induced by Akt and Kras. The latter tumors resembled human gliosarcoma as they were biphasic tumors composed of two distinct histologic parts, one glioblastoma-like and one sarcoma-like (27). Interestingly, Sox10 was virtually absent from the sarcoma-like elements but heavily expressed in the glioblastoma-like regions. The origin of the sarcoma-like elements in gliosarcoma has not been determined, although it is generally believed that the cells, despite their sarcoma-like morphology, have a common origin with the glioblastoma-like cells. In addition, the genetic aberrations in glioblastoma and gliosarcoma indicate that the genomic changes are similar (28). Further studies are required to resolve if loss of Sox10 expression is one of the events that result in a sarcomatous phenotype.

The expression pattern of Sox10 in human was restricted both in normal and tumor tissues. The normal tissues that stained convincingly positive were brain, epidermal melanocytes, and salivary gland. On the tumor tissue microarray, malignant gliomas and all malignant melanomas and scattered cells in basal cell carcinomas expressed Sox10. Sox10 is known to be expressed in melanocytes, and inactivation of one Sox10 allele leads to melanocyte defects, often presented as the Waardenburg syndrome (depigmentation of skin, hair, and iris; refs. 14, 29). We confirmed the findings of Bannykh et al. (30) that Sox10 is expressed in all human glioma subtypes, even in low-grade astrocytomas, compared with the mouse tumors. Despite the fact that the expression of Sox10 in the human adult brain is restricted to oligodendrocytes, Sox10 cannot be used as a marker for oligodendroglioma. The finding of Sox10 expression in a wide range of human gliomas suggests that oligodendroglioma and astrocytoma may have a common precursor. In the adult brain, type B cells, putative neural stem cells that express both Sox2 and GFAP, have been identified in the subventricular zone of the lateral ventricles and in the subgranular layer of hippocampus (19, 31). These type B cells could be the cell of origin for human gliomas and give rise to an immature/progenitor tumor cell population expressing Sox10. Alternatively, human gliomas are derived from bipotential oligodendrocyte precursors, as discussed above with regard to the experimental mouse gliomas. In childhood medulloblastoma and PNET, there was no or little expression of Sox10 compared with the gliomas. The PNET cell of origin is suggested to be an undifferentiated or less differentiated neuroepithelial cell present in any part of the brain (23, 32), and, for at least a subset of medulloblastomas, the cell of origin has been suggested to be granule neuron precursor cells originated from the external granular layer of the cerebellum (22, 32). The differences in tumor cell of origin could be a reason why these tumor types show different Sox10 staining patterns. Further studies will address this important issue.

Materials and Methods

DNA Constructs of RCAS Vectors

A V5 tag was added to the 3′ end of the human SOX10 cDNA clone (originally from Dr. Michael Wegner, Erlangen-Nürnberg University; kindly provided by Dr. Olle Kämpe, Uppsala University, Uppsala, Sweden). The 1,591-bp fragment, which includes the entire coding sequence of SOX10 plus the V5 tag, was subcloned into the RCAS-X vector by digestions with the restriction enzymes ClaI and NotI to construct the RCAS-SOX10 virus vector. The PDGFB-containing vector is RCAS-PDGFB-IRES-EGFP (ref. 33; here referred to as RCAS-PDGFB). RCAS-X is empty vector.

Transfection of DF-1 Cells

Chicken DF-1 fibroblasts were grown in DMEM complemented with 12% FCS. The RCAS-SOX10 and RCAS-X vectors were transfected into DF-1 cells with the FuGENE 6 transfection reagent (Roche). The vectors did replicate within the producer cell populations.

Mice

The generation of the Ntv-a wt mouse line expressing the tv-a receptor behind the nestin promoter has been described earlier (34, 35) as well as the Ntv-a Arf−/− mouse line (35, 36). The brain tumor sections induced by RCAS-Kras and RCAS-Akt have been described earlier (36).

Tumor Surveillance

Neonatal mice were injected in the right cerebral hemisphere with 2 μL of DF-1 cells producing the appropriate RCAS viruses (35). The mice were sacrificed when ill or at 12 weeks of age. Statistical analysis was done with the software GraphPad Prism 4 using the Kaplan-Meier survival analysis.

Human Glioma Tissue Collection

Human glioma tissue sections were obtained from patients diagnosed at the Department of Genetics and Pathology, Uppsala University Hospital, Uppsala, Sweden. The classification of the glioma type and grade were reevaluated by a neuropathologist (T.O.).

Acknowledgments

Footnotes

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